Von Willebrand factor (vWF) is the largest known molecule circulating in plasma. It exists as a series of large disulfide-linked multimers, the basic subunit of which has a molecular weight of about 260 kilodaltons (KDa). The smallest form of vWF in plasma is a dimer of about 440-500 KDa and the largest forms are multimers of the dimer with molecular weights reaching up to 20 million daltons. The assembly of subunits which are linked together may be cell specific, the vWF being synthesized and polymerized in the megacaryocytes and endothelial cells.
This factor plays an essential role in hemostasis through two distinct functions: it transports and stabilizes factor VIII in the blood stream and, as an adhesive protein, it permits the spreading, the attachment and the aggregation of the blood platelets on the vascular subendothelium thus contributing to the swift healing of injured vessels.
A congenital vWF deficiency, or a structural anomaly of this factor, gives rise to von Willebrand disease which initially takes the form of hemorrhages, particularly cutaneous and of the mucous membranes. The clinical forms taken by this disease are very heterogenous and pose major problems in the event of surgery. Treatment of yon Willebrand disease is essential in order to correct primary hemostasis (bleeding time) and coagulation (activated cephalin time and F VIII activity) anomalies.
The disease is treated by substitute therapy with vWF-enriched human plasma derivatives (for example, the cryoprecipitated fraction of plasma or the concentrates of Factor VIII containing a sufficient quantity of vWF). However, these products are not standardized for the treatment of von Willebrand disease. In addition, the poorly purified fractions of blood plasma, especially cryoprecipitate, are not free from the risk of viral contamination because they are often not subjected to any efficient viral inactivation step. Furthermore, they lead to an excess of contaminating proteins which the patient does not need and which can cause immune reactions after multiple injections.
Purified Factor VIII, on the contrary, can be subjected to efficient virus inactivation treatment, but its purification process has been optimized for treating hemophilia A patients and not for vWF deficient-patients. In fact, the recently developed and increasingly effective processes, such as immunoaffinity or ion exchange purification used for preparing Factor VIII, produce concentrates that no longer contain enough vWF to be efficient in the treatment of von Willebrand disease.
It is to meet this need of an efficient way for treating von Willebrand disease that the Applicants have developed a new industrial process for purifying vWF while still obtaining optimum benefit from the isolation of different plasma molecules. In particular, it permits, in one step, the preparation of a concentrate of Factor VIII (according to a process described in EP Application 0 359 593) and to recover a separate vWF fraction from the same batch of cryoprecipitate, thus allowing the optimal use of human plasma. The vWF fraction thus obtained is purified by two additional chromatographic steps which provide a vWF concentrate of very high purity.
The complexity of the vWF molecule makes it very difficult to purify. Small-scale methods, i.e., 5 to 2000 ml for the purposes of analytical study, have already been described (Thorell et al., Thromb. Res. 1984, 35: 431-450), but it has not been possible to adapt these methods for vWF preparation on an industrial scale. In addition, the concept of making the best possible use of cryoprecipitate by producing vWF in addition to FVIII was not considered.
vWF has been purified by differential solubilization on sulfated compounds in the presence of glycine (Berntorp et al., Vox Sang. 1989, 56: 212), sulfated compounds (Winkelman et al., Vox Sang. 1989, 57: 97) and by using different methods of chromatographic separation, such as molecular size exclusion (Perret et al., Haemostasis 1984, 14: 289) and ion exchange (Austen et al., Thromb Haemostas. 1982, 48: 295). However, these techniques give either low yields of vWF or have a low gel capacity, or do not make the simultaneous isolation of FVIII and vWF possible, which make them less convenient for an industrial application.
In addition, Berntorp et al. (Vox Sang. 1989, 56: 212) obtain a vWF of low purity: 45 U Ag/mg protein (p. 213) whereas the Applicants obtain 205 U Ag/mg protein. Similarly, Winkelman et al. (Vox Sang. 1989, 57: 97) obtain 10 U Ag/ml protein (p. 101).
Perret et al. (Haemostasis 1984, 14: 289), perform a defibrination step (to eliminate fibrinogen as fibrin molecules) with the use of calcium as well as enzymes from snake venom. This renders the preparation obviously unsuitable for therapeutic purposes. Moreover, gel filtration systems such as the one used by Perret et al. are hardly compatible with industrial scaling up, since they allow a flow rate of only 10 cm/h or less and show a high risk of plugging, especially in the presence of fibrinogen and fibronectin. Also the purification factor is known to be usually low due to the poor resolution of proteins in this chromatographic system.
Austen et al. (Throm. Hacmostas. 1982, 48: 46) also obtain a low purity concentrate (8 U Ag/mg protein) and relatively low yield, probably due to their drastic chromatographic conditions (pH 5.5).
Harrisson et al. (Thromb. Res. 1988, 50: 295) use dextran sulfate-sepharose as a chromatographic matrix; this material has a low retention capacity for the vWF. As a result, they obtain a vWF preparation of low specific activity: 2-4 U/mg protein (p. 301).
Finally, most of these products contain a rather large proportion of denatured or inactive forms of vWF as evidenced by the ristocetin cofactor activity (RCo)/antigen ratio ranging from 0.08 to 0.8 (Lawrie et al., Br. J. Haematol. 1989, 73: 100). This makes these products less efficient for therapeutic use in von Willebrand disease. On the contrary, the Applicant's procedure allows the recovery of vWF with a RCo/antigen ratio higher than unity, which is comparable to that of native vWF from normal pool plasma.